CN107417744B - Sucrose derivative, preparation method thereof and application of sucrose derivative as anti-cancer drug - Google Patents
Sucrose derivative, preparation method thereof and application of sucrose derivative as anti-cancer drug Download PDFInfo
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Abstract
本发明涉及医药技术领域，具体是从菊科兔耳风属植物云南兔耳风(Ainsliaea yunnanensis Franch.)中分离得到的一类结构新颖的羟基被异戊酸酯化的蔗糖衍生物及其制备方法，以及在制备抗癌药物中的应用。本发明提供了一种蔗糖衍生物，其化学结构如式I所示：本发明的蔗糖衍生物对人肺腺癌细胞A549具有显著细胞毒活性，可用于制备抗癌药物。The invention relates to the technical field of medicine, in particular to a class of sucrose derivatives with a novel structure in which hydroxyl groups are isovalerate esterified from Ainsliaea yunnanensis Franch. The method, and the application in the preparation of anticancer drugs. The present invention provides a kind of sucrose derivative, and its chemical structure is shown in formula I: 
The sucrose derivative of the present invention has significant cytotoxic activity on human lung adenocarcinoma cell A549, and can be used for preparing anticancer drugs.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a sucrose derivative with a novel structure and esterified by isovaleric acid and a preparation method thereof, which is separated from Ainsliaea Yunnanensis of Compositae, and an application thereof in preparing anticancer drugs.
Background
The plants of the lagotis (Ainsliaea DC) of the Compositae family are mainly distributed in southeast Asia, about 70 plants all over the world, 44 varieties and 4 varieties exist in China, and the rest plants are all produced in the Yangtze river basin and the areas of the south province except 1 plant produced in the northeast China (Chinese plant record editting Committee of Chinese academy of sciences, China plant J [ M ]. seventh nineteen volume, Beijing: scientific Press, 1996: 23.). Terpenes, sesquiterpene dimers and sesquiterpene trimers separated from the plant have good cytotoxic activity on part of tumor cells. For example, 5 terpenoids mokko lactone, betalonic acid, betallinic acid, zaluzanin C and glucozazanin C isolated from Ainsliaea fragrans (A. acerifolia) are significantly non-specific against 5 human tumor cell lines (Choi SZ, Yang MC, Choi SU, et al. cytotoxigenic and Lians from the same species of the Ainsliaa acerifolia [ J ]. Arch. Pharm. Res.,2006,29(3):203-208.) isolated from the plant Ainsliaea fragaria (A. macerifolia) and more active against human lung adenocarcinoma cells (C. gonorrhoea A), ovarian adenocarcinoma cells (SK-OV-3), melanoma SK-MEL-2, central nervous system tumor XF498 and human colorectal carcinoma cell HCT15, and human lung adenocarcinoma cells (C. A. mace. gonorrhoea A. and C) than human lung adenocarcinoma cells (C. gonorrhoea A. oviduct) and human adenocarcinoma cells (C. granulosa-9. faecal) isolated from the plant genus Ainsliaea fragilis. The research on the phytochemical components and the pharmacological activity of Lamatoda Lasiosphaera plants progresses [ J ] Chinese medicinal materials, 2012,7(35): 1171-.
The group of subjects to which the present inventors belong has also been devoted to research on plants of this genus, and results of the research have been filed and obtained in chinese patent CN200810035858.6, granted publication No. CN 101318946B; and CN200810035859.0, grant bulletin number CN 101318966B; the two patents relate to dimeric sesquiterpene compounds extracted and separated from Ainsliaea macrocephala, namely Ainsliaea dimer A, Ainsliaea dimer B and Ainsliaea dimer C, which can be used for preparing anti-inflammatory and antitumor drugs. In addition, Chinese patent application CN200810038569.1, granted publication No. CN101284004B, which relates to the extraction and separation of diterpenoids-gochnatiolide A and gochnatiolide B from gochnatiolide, has good inhibitory effect on various human tumor cells and also has significant inhibitory effect on inflammation.
Yunan Lagotis (Ainsliaea yunnanensis Franch.) is a perennial herb of Lagotis of Compositae, is a unique plant in China and is mainly distributed in Yunnan and Guizhou provinces. The plant uses the whole herb as the medicine, is called as the avellan, is called as the copper foot waring, the Chinese clinopodium herb, the bone-knitting one-arrow, and is collected in the Chinese herbal medicine compilation, the Kunming folk herbal medicine and the Chinese herbal medicine, and has pungent taste; bitter; neutral in nature, it is commonly used for dispelling wind-damp, relaxing tendons and bones, reuniting bones, mainly for traumatic injuries, fractures, rheumatic arthralgia and myalgia. At present, the research on the chemical components of Yunnan Laurencia obtusa is less, only a few related reports exist, and compounds such as sesquiterpenes, triterpenes, steroids, flavones and small molecular organic phenolic acids are obtained by separation from the Yunnan Laurencia obtusa (Lijinjie, Wang Ali, Zhen of the institute, and the like, the triterpenes component [ J ] in the Yunnan Laurencia obtusa, the Chinese traditional medicine journal, 2013,38(22): 3918-.
To date, no research report on the activity of the Yunnan lepriasis has been found. Therefore, the chemical components of the Yunnan lepriasis are systematically researched, so that the compound with the anti-tumor activity is extracted and separated from the Yunnan lepriasis.
Disclosure of Invention
The invention aims to separate natural products with novel structures and obvious activity from Yunnan Lagoiter, in particular to sucrose derivatives with hydroxyl groups esterified by isovaleric acid; it is another object of the present invention to provide a process for the preparation of such sucrose derivatives; the third purpose of the invention is to provide the medical application of the sucrose derivative, in particular to the application in preparing anti-tumor drugs.
The inventor obtains a sucrose derivative with hydroxyl group esterified by isovaleric acid in the systematic research of Yunnan Lagotis auricular wind chemical components. In vitro cytotoxic activity experiments prove that the compound has obvious cytotoxic activity on human lung adenocarcinoma cells A549. Further action mechanism research shows that the compounds inhibit the growth of A549 cells by inducing cell cycle arrest and apoptosis, and the induction of A549 cell apoptosis is realized through a mitochondrial pathway and an ROS (reactive oxygen species) pathway. Therefore, the sucrose derivative in the invention has the function of being a potential anti-tumor drug.
The first aspect of the invention provides a sucrose derivative, the chemical structure of which is shown as formula I:
in the formula I, R1～R8The groups are respectively selected from: a hydrogen atom or an isovaleryl group, a substituted or unsubstituted C1-6 straight chain or branched chain acyl group, or a substituted or unsubstituted five-membered or six-membered aromatic ring acyl group;
the number of the acyl groups is 4-5, and the balance is hydrogen atoms (the acyl groups are isovaleryl groups, substituted or unsubstituted C1-6 acyl groups containing straight chains or branched chains, and substituted or unsubstituted acyl groups containing five-membered or six-membered aromatic rings).
In the formula I, R1～R8The groups may be the same or different.
The mother nucleus of the sucrose derivative is a sucrose molecule, and the hydrolysis product of the sucrose derivative is a molecule of D-glucose and a molecule of D-fructose.
The sucrose derivative is a sucrose derivative with hydroxyl groups esterified by isovaleric acid.
In the formula I, the 4-5 acyl groups are formed by esterification reaction of isovaleric acid and hydroxyl on sucrose.
In the formula I, R1～R8The groups are preferably: a hydrogen atom, or isovaleryl group.
Preferred compounds of the invention, R thereof1～R8The combinations of (a) and (b) are as follows:
compound 1: R1＝R2＝R4＝R6＝R7＝-COCH2CH(CH3)2,R3＝R5＝R8＝H；
Compound 2: R1＝R2＝R6＝R7＝R8＝-COCH2CH(CH3)2,R3＝R4＝R5＝H；
Compound 3: R1＝R2＝R3＝R6＝R7＝-COCH2CH(CH3)2,R4＝R5＝R8＝H；
Compound 4: R1＝R3＝R4＝R6＝R7＝-COCH2CH(CH3)2,R2＝R5＝R8＝H；
Compound 5: R1＝R4＝R6＝R7＝-COCH2CH(CH3)2,R2＝R3＝R5＝R8＝H；
Compound 6: R1＝R2＝R4＝R7＝-COCH2CH(CH3)2,R3＝R5＝R6＝R8＝H；
Compound 7: R1＝R2＝R6＝R7＝-COCH2CH(CH3)2,R3＝R4＝R5＝R8＝H；
Compound 8: R1＝R2＝R4＝R6＝-COCH2CH(CH3)2,R3＝R5＝R7＝R8＝H。
In a preferred embodiment of the invention, compound 2: 1 ', 4, 4', 6,6 '-pentakis-O- (3-methylbutanoyl) - β -D-fructofuranosyl α -D-glucopyranoside, designated ainslioside B (Lagotu's ear sugar B), is the most preferred compound of the invention.
The above compounds 1-8 can be obtained by separating from Ainsliaea Yunnanensis, or by chemical synthesis.
The second aspect of the invention provides a preparation method of the sucrose derivatives with hydroxyl groups esterified by isovaleric acid, in particular to a method for separating and preparing the sucrose derivatives from Ainsliaea Yunnanensis belonging to Ainsliaea.
The method of the invention comprises the following steps:
the preparation method comprises the steps of crushing dry whole grass of Yunnanensis (Ainsliaea yunnanensis Franch.) of Ainsliaea, heating and refluxing the crushed whole grass with ethanol, dispersing extract obtained after decompression and concentration of extract with water, preparing extraction parts with four different polarities of petroleum ether, trichloromethane, ethyl acetate and n-butyl alcohol by adopting a liquid-liquid extraction method respectively, taking common chromatographic fillers such as silica gel, macroporous adsorption resin, ODS, Sephadex LH-20 and the like as stationary phases for one time or multiple times, taking common solvents such as petroleum ether, trichloromethane, ethyl acetate, acetone, methanol, acetonitrile, water and the like as an eluent or a recrystallization solvent in a single or different combination ratio, and separating and purifying to obtain the sucrose derivative with hydroxyl groups esterified by isovaleric acid.
In the method, preferably, 95 percent and 80 percent of ethanol are respectively used for reflux extraction twice, the extracting solutions are combined, and the temperature of heating reflux extraction is 80 ℃.
The liquid-liquid extraction method is a conventional extraction method.
The petroleum ether extraction part is subjected to gradient elution by adopting a petroleum ether-ethyl acetate system (200: 1-0: 1).
The chloroform extraction part is subjected to gradient elution by adopting chloroform-methanol (100: 0-0: 1).
The ethyl acetate extraction part is subjected to gradient elution by adopting petroleum ether-ethyl acetate (100: 1-0: 1).
The n-butanol extraction part is subjected to gradient elution by adopting chloroform-methanol (100: 1-0: 1).
The eluent is preferably chloroform-methanol 50: 1.
The preparation method of the compounds 1-8 of the invention comprises the following steps: pulverizing dried whole plant of Ainsliaea Yunnanensis (15.0kg), extracting with 95% and 80% ethanol under reflux twice, and mixing extractive solutions; carrying out water dispersion on the obtained extract after the extract is subjected to reduced pressure concentration, and carrying out liquid-liquid extraction on the water-dispersed extract sequentially according to the sequence of small to large polarities by adopting petroleum ether, trichloromethane, ethyl acetate and n-butanol to respectively obtain four extraction parts with different polarities, namely petroleum ether, trichloromethane, ethyl acetate and n-butanol; collecting extract (400g) of ethyl acetate part, subjecting to 80-100 mesh silica gel column chromatography, and purifying with petroleum ether-ethyl acetateThe system 100:1,50:1,30:1,20:1,10:1,5:1,0:1 was eluted, and 7 fractions were obtained according to the difference of elution gradient: a (36G; 100:1), B (46G; 50:1), C (58G; 30:1), D (43G; 20:1), E (57G; 10:1), F (49G; 5:1), G (37G; 0: 1); the component C is obtained by gradient elution of petroleum ether-ethyl acetate system 30:1, and the component is subjected to medium-low pressure reverse phase column chromatography and MeOH-H240-50% of O; 50-60 percent; 60 to 70 percent; 70-80%; 80-90%; 90-100%; the difference in the elution gradient of 100% gives 7 fractions: c-1(3 g; 40-50%), C-2(15 g; 50-60%), C-3(6 g; 60-70%), C-4(2 g; 70-80%), C-5(7 g; 80-90%), C-6(6 g; 90-100%), C-7(5 g; 100%); the component C-4 is subjected to 200-mesh 300-mesh silica gel column chromatography, and chloroform-methanol is 200: 1; 100: 1; 50: 1; 30: 1; 10: 1; 5:1 gradient elution separation, wherein chloroform-methanol 50:1 gradient elution fraction (300mg) is purified by Sephadex LH-20 to obtain compound 3(21.0 mg; chloroform-methanol 20:1), and chloroform-methanol 30:1 gradient elution is carried out to obtain 2(12.0 mg; chloroform-methanol 20:1) and 4(7.0 mg; chloroform-methanol 20: 1); c-2 by RP-MPLC with MeOH-H240-50% of O; 50-60 percent; 60 to 70 percent; 70-80%; the gradient elution with 80-100% was carried out by varying the elution gradient to obtain 5 fractions C-21(2.3 g; 40-50%), C-22(1.8 g; 50-60%), C-23(2.9 g; 60-70%), C-24(2.3 g; 70-80%), C-25(3.1 g; 80-100%); the component C-21 is subjected to 200-mesh 300-mesh silica gel column chromatography, and petroleum ether-ethyl acetate 50: 1; 40: 1; 30: 1; 20: 1; 10: 1; gradient elution 1:1, wherein gradient elution of petroleum ether-ethyl acetate 20:1 gives compounds 5(9.3 mg; chloroform-methanol 20:1) and 7(7.1 mg; chloroform-methanol 20:1), gradient elution of petroleum ether-ethyl acetate 10:1 gives compound 6(11.2 mg; chloroform-methanol 20:1), gradient elution of petroleum ether-ethyl acetate 1:1 gives compound 8(6.4 mg; chloroform-methanol 20: 1); fraction C-23 via RP-MPLC with MeOH-H240-50% of O; 50-60 percent; 60 to 70 percent; 70-80%; 80-90%; 90-100%; elution with a 100% gradient, with a 70-80% MeOH gradient, provided Compound 1(10.5 mg; chloroform-methanol 20: 1).
In a third aspect of the present invention, an application of the sucrose derivative with hydroxyl group esterified by isovaleric acid in preparing an antitumor drug is provided.
The tumor includes but is not limited to lung adenocarcinoma, colon cancer, breast cancer, liver cancer and the like.
The sucrose derivatives with hydroxyl groups esterified by isovaleric acid refer to compounds with significant cytotoxic activity to human lung adenocarcinoma cells A549.
The drug is a sucrose derivative with hydroxyl group esterified by isovaleric acid and a pharmaceutically acceptable medicinal composition thereof, or a medicinal composition which is composed of the sucrose derivative and the pharmaceutically acceptable medicinal composition as active ingredients and comprises a medicinal carrier combination for preparing anti-tumor related drugs; can be administered orally, parenterally, directly in brain, through nasal cavity and brain targeting, by genetic engineering, by receptor mediated transport or by other local routes; the administration dosage form can be pharmaceutically acceptable dosage forms such as tablet, dispersible tablet, buccal tablet, orally disintegrating tablet, sustained release tablet, granule, pill, capsule, emulsion, solution, suspension, injection, instillation, powder injection or aerosol, etc.
The invention has the beneficial effects and meanings that: the active ingredients of the traditional Chinese medicine are obtained from Yunnan lepriasis, the traditional Chinese medicine has a large amount of medical history in China, resources are very rich, and the lepriasis which belongs to the same genus plant is artificially cultured. The research finds that the traditional Chinese medicine composition can be used as a potential anti-tumor medicine, develops a new treatment field of the traditional Chinese medicine, and has very important significance for moving the development of economy in Yunnan areas.
Drawings
FIG. 1 shows the structure of Compound 2 and its effect on the inhibition of A549 cell growth; wherein (A) the structure of Compound 2; (B) the compound 2 with the concentration of 1 mu M,3 mu M and 10 mu M has the inhibition rate on A549 cells after 24,48 and 72 hours; (C) the change of the nuclear morphology of A549 cells after 24 hours of the action of compound 2 with different concentrations;
FIG. 2 shows that Compound 2 is able to induce cell G1Phase retardation; the experimental data are presented as mean ± sd using One-Way analysis of variance (One-Way ANOVA), Duncan test, P<0.05 is significantly different (, P)<0.05vs.Vehicle)；
FIG. 3 is a graph of the induction of apoptosis in A549 cells by different doses of Compound 2; wherein (A) representative pictures of apoptosis after treatment of A549 cells with different concentrations of Compound 2, wherein the X-and Y-axes represent Annexin V-FITC and PI staining, respectively; (B) apoptosis trend statistics of A549 cells; the experimental data are all expressed as mean ± standard error, with One-Way ANOVA, Duncan test, P <0.05 is significantly different (.;, P < 0.05;. P <0.01vs. vehicle);
FIG. 4 is a graph of the effect of varying concentrations of Compound 2 on mitochondrial membrane potential in A549 cells; wherein (A) mitochondrial membrane potential flow diagrams of A549 cells after the action of different concentrations of Compound 2; (B) loss of mitochondrial membrane potential in a549 cells after the action of compound 2 at different concentrations; the experimental data are expressed as mean ± standard error, and the difference is significant when P <0.05 (x, P <0.05vs. vehicle) is detected by using One-way analysis of variance (One-WayANOVA) and Duncan test;
FIG. 5 shows the ROS content in A549 cells after the action of different concentrations of Compound 2; wherein (A) DCFH-DA fluorescence intensity is detected by a flow cytometer; (B) the active oxygen ROS content in A549 cells.
Detailed Description
The invention will now be further described with reference to examples, but the practice of the invention is not limited thereto.
Example 1: preparation of anslide B (2)
1 sample origin
Whole plant of Ainsliaea yunnanensis Franch (Ainsliaea yunnanensis Franch.) of Ainsliaea of Compositae.
2 extraction separation and purification method
Pulverizing dried whole plant of Ainsliaea Yunnanensis (15.0kg), extracting with 80L 95% and 80% ethanol under reflux twice, and mixing extractive solutions; concentrating the extract under reduced pressure to obtain extract, dispersing in water, and sequentially performing liquid-liquid extraction on the water-dispersed extract with petroleum ether (15L), chloroform (15L), ethyl acetate (15L) and n-butanol (15L) according to the sequence of polarity from small to large to respectively obtain four extraction parts with different polarities, namely petroleum ether, chloroform, ethyl acetate and n-butanol; getSubjecting the extract (400g) of ethyl acetate part to 80-100 mesh silica gel column chromatography, eluting with petroleum ether-ethyl acetate system 100:1,50:1,30:1,20:1,10:1,5:1,0:1 to obtain 7 components according to different elution gradients: a (36G; 100:1), B (46G; 50:1), C (58G; 30:1), D (43G; 20:1), E (57G; 10:1), F (49G; 5:1), G (37G; 0: 1); the component C is obtained by gradient elution of petroleum ether-ethyl acetate system 30:1, and the component is subjected to medium-low pressure reverse phase column chromatography and MeOH-H240-50% of O; 50-60 percent; 60 to 70 percent; 70-80%; 80-90%; 90-100%; the difference in the elution gradient of 100% gives 7 fractions: c-1(3 g; 40-50%), C-2(15 g; 50-60%), C-3(6 g; 60-70%), C-4(2 g; 70-80%), C-5(7 g; 80-90%), C-6(6 g; 90-100%), C-7(5 g; 100%); the component C-4 is subjected to 200-mesh 300-mesh silica gel column chromatography, and chloroform-methanol is 200: 1; 100: 1; 50: 1; 30: 1; 10: 1; gradient elution separation is carried out at a ratio of 5:1, wherein gradient elution of chloroform-methanol is carried out at a ratio of 30:1 to obtain a compound 2(12.0 mg; chloroform-methanol is carried out at a ratio of 20: 1).
3 structural characterization of Compound 2
With modern spectroscopic techniques (IR,1H-NMR,13C-NMR,DEPT,1H-1h COSY, HSQC, HMBC, NOESY, HRESIMS) to identify the structure of compound 2, as shown in formula II:
anslide B (2) as a colorless oil; IR (KBr) vmax3446,2960,2929,2871,1741,1662,1468,1371,1296,1252,1188,1095,1011cm-1；HRESIMS(positive)m/z785.3952[M+Na]+(calcd 785.3936)；1H-NMR(500MHz,CDCl3)δ:5.55(1H,d,J＝3.5Hz,H-1),5.16(1H,d,J＝7.0Hz,H-4'),4.87(1H,t,J＝9.5Hz,H-4),4.32(2H,m,H-1'),4.31(2H,m,H-6'),4.25(1H,t,J＝9.5Hz,H-3'),4.19(1H,m,H-5),4.15(1H,m,H-6),4.08(1H,m,H-5'),3.88(1H,t,J＝9.5Hz,H-3),3.66(1H,m,H-2),2.21-2.26(10H,m,CH2×5),2.06-2.14(5H,m,CH×5),0.92-0.98(30H,m,CH3×10)；13C-NMR(125MHz,CDCl3)δ:173.0(C＝O),172.9(C＝O),172.7(C＝O),172.6(C＝O),172.2(C＝O),104.3(C-2'),92.3(C-1),78.0(C-5'),77.4(C-4'),77.2(C-3'),72.3(C-2),72.2(C-3),70.2(C-4),68.9(C-5),64.1(C-6'),63.0(C-1'),62.1(C-6),42.7-43.2(CH2×5),25.4-25.7(CH×5),22.3(CH3×10).
Example 2: determination of the Glycogenin and sugar Structure in Compound 2
Compound 26.0 mg dissolved in 5% KOH-H2In O (5mL), heated to 95 ℃ under reflux for 3 h. The reaction mixture was acidified with hydrochloric acid to PH 4.0, extracted three times with chloroform and n-butanol, respectively, and dried under reduced pressure to obtain a chloroform layer 2.1 mg. After dissolving with deuterated chloroform, the obtained product is identified as isovaleric acid by NMR spectrum measurement.
Isovaleric acid:1H NMR(in CDCl3,500MHz)δH2.23(2H,d,J＝7.5Hz),2.12(1H,m),0.99(6H,d,J＝7.0Hz)；13C NMRδC178.8(-COOH),43.0(CH2),25.5(CH),23.3(CH3),22.3(CH3).
Dissolving n-butanol layer of alkaline hydrolysate of Compound 2 in 2M HCl-H with a small amount of methanol2In O (5mL), heated at 95 ℃ under reflux for 4h, extracted three times with n-butanol, the aqueous layer was dried under reduced pressure and then dissolved in water and directly analyzed by HPLC with an ELSD detector: YMC-Pack NH2Analyzing chromatographic column (5 μm,12nm,4.6 × 250mm), gradient eluting with 75% acetonitrile-water, flow rate of 1mL/min, sample amount of 3 μ L, comparing with sugar standard, determining sugar unit composition, D-glucose [ t [ t ] ]R＝16.308min,[α]23 D+53(c0.113,H2O)]D-fructose [ t ]R＝13.116min),[α]23 D-149(c 0.118,H2O)]。
Example 3: cytotoxic Activity assay
1. Experimental Material
Human lung adenocarcinoma cells (a549) were purchased from the institute of biochemistry and cell biology, shanghai institute of life science, china academy of sciences;
compound anslide B (2), prepared in example 1.
2. Cytotoxic activity screening and action mechanism research method
(1) MTT method for detecting cytotoxic activity
Collecting A549 cells growing in logarithmic phase, and adjusting cell suspension concentration to 1 × 105After one well, the cells were inoculated in a 96-well plate with 100. mu.L/well volume, 37 ℃ and 5% CO2Culturing under the condition, adding the compounds to be tested with different concentrations after the cells adhere to the wall, and continuing culturing for 24 h. After the termination of the incubation, 10. mu.L of 5mg/mL MTT solution was added to each well, the incubation was continued for 4 hours, the culture medium was centrifuged and discarded, 100. mu.L DMSO was added to each well to dissolve the formazan product formed, and the absorbance of each well at 570nm was measured using a plate reader.
(2) Time-dose dependent assay of cytostatic rate
MTT method: the cultured A549 cells were digested with trypsin and then digested at 6X 103The cells were seeded in 96-well plates at a concentration of one/mL, 100. mu.L of cell suspension was added to each well, and the mixture was incubated at 37 ℃ with 5% CO2After 24h in the incubator, the prepared test sample solution anslide B (1,3,10 μ M), DMSO (0.1%) in the blank, 10 μ L/well, and three multiple wells were set and placed at 37 ℃ and 5% CO2After the culture box acts for 24 hours, 48 hours and 72 hours. Adding 10 mu L of 5mg/mL MTT solution into each hole, acting at 37 ℃ for 4h, removing supernatant, adding 100 mu L/hole DMSO, placing in an incubator, and detecting OD value at 570nm wavelength by using a full-automatic enzyme standard instrument after dissolution. The inhibition ratio and IC were calculated according to the following formulas50: the inhibition ratio (%) (control OD value-experimental OD value)/control OD value × 100%. Then, the concentration of the compound to be added was plotted on the abscissa and the inhibition (%) was plotted on the ordinate, and the IC was calculated by a direct graphical method50。
(3) Apoptosis detection
The condition of apoptosis is detected by Annexin V-FITC/PI double staining. The cultured A549 cells were trypsinized and then treated at 2X 105The cells were seeded in 6-well plates at a concentration of 2mL per well and placed at 37 ℃ in 5% CO2After 24h in the incubator, the prepared test sample solution anslide B (1,3, 10. mu.M) was added to the experimental group, doxorubicin was added at 10. mu.M to the positive control group, and DMSO (0.1%) was added to the blank group at 37 ℃ and 5% CO2After 24 hours of action in the incubator, the cells were collected and placed in a centrifuge tube and washed 2 times with PBS (containing 2% FBS) (2000r/min centrifugation 5 m)in), the liquid was removed as much as possible, 200. mu.L binding buffer suspension cells were added, and 5. mu.L annexin V (10. mu.g/mL) and 10. mu.L PI (20. mu.g/mL) were added in this order, and the mixture was left in the dark at room temperature for 10min and then immediately subjected to flow cytometry.
(4) Detection of apoptosis by Hoechst 33258 staining method
The condition of apoptosis was detected by Hoechst 33258 staining. The cultured A549 cells are digested by pancreatin and then inoculated in a 6-well plate, 2mL of cell suspension is added into each well, and the mixture is placed at 37 ℃ and 5% CO2After 24h in the incubator, the test sample solution anslide B (1,3, 10. mu.M) was added to the experimental group, 10. mu.M doxorubicin was added to the positive control group, and DMSO (0.1%) was added to the blank group at 37 ℃ and 5% CO2After 24 hours of action in the incubator, freshly prepared 4% paraformaldehyde is added and fixed for 15min at room temperature, PBS is added for washing for 2 times, Hoechst 33258(50 mu g/mL) is added and is placed in a dark place at 37 ℃ for dyeing for 30min, PBS is used for washing, cells are suspended, and the shape of cell nuclei is analyzed by a fluorescence microscope.
(5) Cell cycle assay
Cell cycle detection kits were used to detect cell cycle conditions. The cultured A549 cells are digested by pancreatin and then inoculated in a 6-well plate, 2mL of cell suspension is added into each well, and the mixture is placed at 37 ℃ and 5% CO2After 24h in the incubator, the prepared test sample solution anslide B (1,3, 10. mu.M) was added to the experimental group, doxorubicin was added at 10. mu.M to the positive control group, and DMSO (0.1%) was added to the blank group at 37 ℃ and 5% CO2After 24h in the incubator, the collected cells were placed in a centrifuge tube, washed 2 times with PBS (containing 2% FBS) (5 min centrifugation at 2000 r/min), drained as much as possible, added cold absolute ethanol overnight at 4 ℃, washed 2 times with PBS, added 1mL of PI staining solution (containing 3.8mM sodium citrate and 50. mu.g/mL PI), added 50. mu.L of RNase A solution (100. mu.g/mL RNase A), and immediately placed in the dark at room temperature for 30min, the DNA content of each cell cycle was measured using a flow cytometer.
(6) Mitochondrial membrane potential detection
And (3) adopting a mitochondrial membrane potential detection kit to detect the condition of the mitochondrial membrane potential. The cultured A549 cells are digested by pancreatin and then inoculated on a 6-well plate2mL of cell suspension was added to each well, and the mixture was incubated at 37 ℃ with 5% CO2After 24h in the incubator, the prepared test sample solution anslide B (1,3, 10. mu.M) was added to the experimental group, doxorubicin was added at 10. mu.M to the positive control group, and DMSO (0.1%) was added to the blank group at 37 ℃ and 5% CO2After 24 hours of action in the incubator, 10 μ M Rhodamine 123(Rhodamine 123) was added and the mixture was stained in the dark at room temperature for 30min, and the cells were washed with PBS and suspended immediately followed by detection with a flow cytometer.
(7) Intracellular reactive oxygen species detection
And detecting the content of the ROS in the cells by adopting a Reactive Oxygen Species (ROS) detection kit. The cultured A549 cells are digested by pancreatin and then inoculated in a 6-well plate, 2mL of cell suspension is added into each well, and the mixture is placed at 37 ℃ and 5% CO2After 24h in the incubator, the prepared test sample solution anslide B (1,3, 10. mu.M) was added to the experimental group, doxorubicin was added at 10mM to the positive control group, and DMSO (0.1%) was added to the blank group at 37 ℃ and 5% CO2After 24 hours of action in the incubator, the cells were collected, washed 2 times with PBS, the cells were suspended in DMEM medium (containing 10. mu.M 2',7' dichlorohydrofluorescent ethylene glycol) and then placed at 37 ℃ for 20min, and the cells were immediately detected by flow cytometry after being washed 2 times with PBS.
3. Results of the experiment
The IC of the cytotoxic activity of the compound 2 on A549 human lung adenocarcinoma cells can be known from the result of the cytotoxic activity test50The value was 3.3. mu.M, showing significant cytotoxic activity. (the positive control drug is adriamycin, IC)50Value of 0.034. mu.M)
The MTT method is adopted to test the cytotoxic activity of the compound 2 on human lung adenocarcinoma cells (A549), and the A549 cells are taken as a model to research the action mechanism of the compound 2 for inhibiting the growth of the lung adenocarcinoma cells. Fig. 1B shows that compound 2 has a concentration-time dependence on inhibition of a549 cell growth.
After the compound 2 with different dosages of 1,3 and 10 mu M acts on the A549 cells for 24h, the survival rate of the A549 cells is obviously reduced, the influence of the compound 2 on the nuclear morphology of the A549 cells is tested by adopting a Hoechst 33258 staining method, and a plurality of A549 cells with different concentrations of the compound 2 act for 24h and undergo nuclear morphology change (figure 1C). The above experimental results further indicate that compound 2 can inhibit tumor cell growth dose-dependently.
The effect of compound 2 on the cell growth cycle was examined by flow cytometry after PI staining. After 24h of treatment of A549 cells with different doses of Compound 2 as shown in FIG. 2, Compound 2 was able to induce G dose-dependently1Increase in the proportion of cells in phase S, G2Reduction in the proportion of cells in phase. The experimental results show that the compound 2 can induce A549 cell cycle arrest at G1And (4) period.
And detecting whether the compound 2 can induce apoptosis by adopting Annexin V-FITC/PI double staining. Apoptosis was observed in a549 cells treated with compound 2 at various concentrations for 24h as shown in fig. 3A and 3B, and was more pronounced with increasing compound concentration.
The effect of compound 2 on mitochondrial membrane potential changes was analyzed by flow cytometry after staining with Rhodamine 123(Rhodamine 123). After 24h of treatment of a549 cells with different doses of compound 2, the mitochondrial membrane potential of a549 cells decreased with increasing compound dose (fig. 4). The above experimental results indicate that compound 2 may induce apoptosis via the mitochondrial pathway.
The effect of compound 2 on intracellular reactive oxygen species ROS content was tested. After the A549 cells are treated for 24 hours by different doses of the compound 2, the DCFH-DA staining is immediately carried out by detecting the DCFH-DA fluorescence intensity by a flow cytometer. Compound 2 was able to increase the reactive oxygen species ROS content in a549 cells as shown in figure 5. The above experimental results indicate that compound 2 may induce apoptosis of a549 cells through ROS pathway.
The results of in vitro cell experiments show that Compound 2 passes through cell G1Phase arrest and apoptosis induction exert inhibitory effects on the growth of a549 cells. In addition, induction of apoptosis is associated with a decrease in Mitochondrial Membrane Potential (MMP) and an increase in intracellular Reactive Oxygen Species (ROS) content.
EXAMPLE 4 Ansloside B tablet preparation
Ansloside B 10g
Lactose 130g
Corn starch 55g
Magnesium stearate 5g
The preparation method comprises the following steps: anslide B, lactose and corn starch were mixed, uniformly moistened with water, the moistened mixture was sieved and dried, sieved again, magnesium stearate was added, and the mixture was tabletted to a weight of 200mg per tablet with an anslide B content of 10 mg.
Example 5 preparation of Ansloside B injection
Ansloside B 2g
Glucose 50g
The preparation method comprises the following steps: anslide B and glucose were dissolved in an appropriate amount of water for injection, and the resulting solution was filtered and aseptically filled into infusion bottles (100 mL per bottle) containing anslide B2 mg per bottle.
Example 6 preparation of lyophilized powder for injection of anslide B
Ansloside B 2g
Mannitol 28g
The preparation method comprises the following steps: anslide B and mannitol were dissolved in an appropriate amount of water for injection, the resulting solution was filtered, aseptically filled into penicillin bottles (10mL penicillin bottles, 2mL each), and lyophilized, each containing anslide B2 mg.
Example 7 preparation of Compounds 1, 3-8
Pulverizing dried whole plant of Ainsliaea Yunnanensis (15.0kg), extracting with 95% and 80% ethanol under reflux twice, and mixing extractive solutions; carrying out water dispersion on the obtained extract after the extract is subjected to reduced pressure concentration, and carrying out liquid-liquid extraction on the water-dispersed extract sequentially according to the sequence of small to large polarities by adopting petroleum ether, trichloromethane, ethyl acetate and n-butanol to respectively obtain four extraction parts with different polarities, namely petroleum ether, trichloromethane, ethyl acetate and n-butanol; taking extract (400g) of the ethyl acetate part, carrying out 80-100-mesh silica gel column chromatography, eluting by a petroleum ether-ethyl acetate system 100:1,50:1,30:1,20:1,10:1,5:1,0:1, and obtaining 7 components according to different elution gradients: a (36G; 100:1), B (46G; 50:1), C (58G; 30:1), D (43G; 20:1), E (57G; 10:1), F (49G; 5:1), G (37G; 0: 1); the component C is obtained by gradient elution of petroleum ether-ethyl acetate system 30:1, and the component is subjected to medium-low pressure reverse phase column chromatography and MeOH-H2O 40 to 50 percent; 50-60 percent; 60 to 70 percent; 70-80%; 80-90%; 90-100%; the difference in the elution gradient of 100% gives 7 fractions: c-1(3 g; 40-50%), C-2(15 g; 50-60%), C-3(6 g; 60-70%), C-4(2 g; 70-80%), C-5(7 g; 80-90%), C-6(6 g; 90-100%), C-7(5 g; 100%); the component C-4 is subjected to 200-mesh 300-mesh silica gel column chromatography, and chloroform-methanol is 200: 1; 100: 1; 50: 1; 30: 1; 10: 1; 5:1 gradient elution separation, wherein chloroform-methanol 50:1 gradient elution fraction (300mg) is purified by Sephadex LH-20 to obtain compound 3(21.0 mg; chloroform-methanol 20:1), and chloroform-methanol 30:1 gradient elution is carried out to obtain 4(7.0 mg; chloroform-methanol 20: 1); c-2 by RP-MPLC with MeOH-H240-50% of O; 50-60 percent; 60 to 70 percent; 70-80%; the gradient elution with 80-100% was carried out by varying the elution gradient to obtain 5 fractions C-21(2.3 g; 40-50%), C-22(1.8 g; 50-60%), C-23(2.9 g; 60-70%), C-24(2.3 g; 70-80%), C-25(3.1 g; 80-100%); the component C-21 is subjected to 200-mesh 300-mesh silica gel column chromatography, and petroleum ether-ethyl acetate 50: 1; 40: 1; 30: 1; 20: 1; 10: 1; gradient elution 1:1, wherein gradient elution of petroleum ether-ethyl acetate 20:1 gives compounds 5(9.3 mg; chloroform-methanol 20:1) and 7(7.1 mg; chloroform-methanol 20:1), gradient elution of petroleum ether-ethyl acetate 10:1 gives compound 6(11.2 mg; chloroform-methanol 20:1), gradient elution of petroleum ether-ethyl acetate 1:1 gives compound 8(6.4 mg; chloroform-methanol 20: 1); fraction C-23 via RP-MPLC with MeOH-H240-50% of O; 50-60 percent; 60 to 70 percent; 70-80%; 80-90%; 90-100%; elution with a 100% gradient, with a 70-80% MeOH gradient, provided Compound 1(10.5 mg; chloroform-methanol 20: 1).
Example 8 structural characterization of Compounds 1, 3-8
With modern spectroscopic techniques (IR,1H-NMR,13C-NMR,DEPT,1H-1h COSY, HSQC, HMBC, NOESY, HRESIMS) identified the structures of compounds 1, 3-8 as follows:
ainsloside A (1) as a colorless oil; IR (KBr) vmax3471,2960,2927,2873,1743,1658,1468,1369,1296,1254,1188,1120,1014cm-1；1H and13c NMR spectral data, see tables 1 and 2; HRESIMS (positive) M/z 785.3939[ M + Na ]]+(calcd 785.3936).
Ainsloside C (3) as colorless oil; IR (KBr) vmax3485,3259,2960,2873,1741,1468,1369,1296,1188,1115,1005cm-1；1H and13c NMR spectral data, see tables 1 and 2; HRESIMS (positive) M/z785.3950[ M + Na ]]+(calcd 785.3936).
Ainsloside D (4) as a colorless oil; IR (KBr) vmax3477,2960,2933,2873,1741,1668,1468,1369,1296,1254,1188,1120,1005cm-1；1H and13c NMR spectral data, see tables 1 and 2; HRESIMS (positive) M/z 785.3942[ M + Na ]]+(calcd 785.3936).
Ainsloside E (5) as colorless oil; IR (KBr) vmax3467,2960,2933,2873,1739,1468,1369,1296,1255,1190,1120,1063,1005cm-1；1H and13c NMR spectral data, see tables 1 and 2; HRESIMS (positive) M/z 701.3365[ M + Na ]]+(calcd 701.3360).
Ainsloside F (6) as a colorless oil; IR (KBr) vmax3460,2960,2931,2873,1741,1468,1371,1296,1190,1099,1012cm-1；1H and13c NMR spectral data, see tables 1 and 2; HRESIMS (positive) M/z701.3362[ M + Na ]]+(calcd 701.3360).
Ainsloside G (7) as a colorless oil; IR (KBr) vmax3408,2960,2933,2873,1743,1468,1369,1296,1254,1188,1093,1003cm-1；1H and13c NMR spectral data, see tables 1 and 2; HRESIMS (positive) M/z 701.3371[ M + Na ]]+(calcd 701.3360).
Ainsloside H (8) as a colorless oil; IR (KBr) vmax3456,2960,2929,2873,1743,1668,1468,1369,1296,1254,1188,1101,1065,1012cm-1；1H and13c NMR spectral data, see tables 1 and 2; HRESIMS (positive) M/z 701.3357[ M + Na ]]+(calcd 701.3360).
TABLE 1 preparation of compounds 1, 3-81H NMR (500MHz) data (in ppm) (CDCl)3)
TABLE 2 preparation of compounds 1, 3-813C NMR (125MHz) data (in ppm) (CDCl)3)
Example 9 cytotoxic Activity assay of Compounds 1, 3-8
The MTT method is adopted to test the cytotoxic activity of the compounds 1 and 3-8 on A549 human lung adenocarcinoma cells, and the experimental material and the experimental method are the same as those in example 3.
The experimental results are as follows:
IC of compound 1 on cytotoxic activity of A549 human lung adenocarcinoma cells50The value was 9.74. mu.M,
IC of compound 3 on cytotoxic activity of A549 human lung adenocarcinoma cells50Has a value of>100μM，
IC of compound 4 on cytotoxic activity of A549 human lung adenocarcinoma cells50The value was 19.93. mu.M,
IC of compound 5 on cytotoxic activity of A549 human lung adenocarcinoma cells50The value was 10.75. mu.M,
IC of compound 6 on cytotoxic activity of A549 human lung adenocarcinoma cells50The value was 60.06. mu.M,
IC of compound 7 on cytotoxic activity of A549 human lung adenocarcinoma cells50The value was 46.19. mu.M,
IC of Compound 8 on cytotoxic Activity of A549 human Lung adenocarcinoma cells50The value was 66.00. mu.M.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
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